The primary focus of the work described in this proposal is the definition of molecular mechanisms of transcriptional specificity within the E2F family of transcription factors. A large body of work has now demonstrated the critical role of E2F proteins in cell cycle progression, particularly playing a key role in the control of expression of genes critical for the transition from G1 into S phase. Moreover, other work has demonstrated the role of E2F proteins in signaling cell fate determinations, particularly the induction of apoptosis in p53-dependent and p53-independent pathways. Other studies suggest that individual members of the E2F family play different roles in these processes, likely through the ability to control the expression of different sets of target genes. Our recent work points to a mechanism for promoter specificity that involves the coordinated action of multiple transcription factors, such that combinations of interactions involving E2F proteins underlies the specificity of promoter recognition. We now propose to extend these initial studies to more globally define the role of E2F protein interactions in the determination of functional specificity. This will include studies to identify additional partners for specific E2F proteins that impart specificity of function. In addition, we will carry out studies to delineate the protein domains and promoter architecture critical for synergy of transcription control by E2F proteins. Finally, we will also employ RNAi molecules that target individual E2F proteins as well as transcriptional partners to establish the functional role of these interactions in mediating E2F function. While it is clear from many experiments that individual E2F proteins are responsible for distinct functional events, the molecular mechanisms that underlie this specificity of function are still largely unknown. We believe the approach described here will provide important insights into these events. Given the pervasive role of Rb pathway mutations in human cancer, understanding the specificity of function in the pathway, particularly the action of the E2F proteins, is critical in considering the development of cancer therapeutics that might target the most relevant aspects of Rb function.